The present invention relates to recombinant DNA which encodes the ApaLI restriction endonuclease and modification methylase, and the production of ApaLI restriction endonuclease from the recombinant DNA.
Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria. When they are purified away from other bacterial components, restriction endonucleases can be used in the laboratory to cleave DNA molecules into precise fragments for molecular cloning and gene characterization.
Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the "recognition sequence") along with the DNA molecule. Once bound, they cleave the molecule within, or to one side of, the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over one hundred and ninety restriction endonucleases with unique specificities have been identified among the many hundred of bacterial species that have been examined to-date.
Bacteria tend to possess at most, only a small number of restriction endonucleases per species. The endonucleases typically are named according to the bacteria from which they are derived. Thus, the species Deinococcus radiophilus for example, synthesizes three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the DNA sequences TTTAAA (SEQ ID NO:1), PuGGNCCPy (SEQ ID NOP:2) and CACNNNGTG (SEQ ID NO:3), respectively. Escherichia coli TY13, on the other hand, synthesizes only one enzyme, EcoRI, which recognizes the DNA sequence GAATTC (SEQ ID NO:4).
It is thought that in nature, restriction endonucleases play a protective role in the welfare of the bacterial cell. They enable bacteria to resist invasion by viruses and foreign DNA. They impart this resist by cleaving the DNA of the invading organism. The cleavage that takes place disables many of the infecting genes and renders the DNA susceptible to further degradation by non-specific nucleases.
A second component of bacterial protective systems are the modification methylases. These enzymes are complementary to restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign, infecting DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one or more of the nucleotides within the sequence by the addition of a methyl group. Following methylation, the recognition sequence is no longer cleaved by the restriction endonuclease. The DNA of a bacterial cell is always fully modified by virtue of the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonucleases. It is only unmodified, and therefore identifiably foreign DNA, that is sensitive to restriction endonuclease recognition and cleavage.
With the advent of genetic engineering technology, it is now possible to clone genes and to produce the proteins and enzymes that they encode in great quantities. The key to cloning restriction endonuclease genes is to develop a simple and reliable method to identify such clones within complex "libraries", i.e., populations of clones derived by "shotgun" procedures, when they occur at frequencies as low as 10.sup.-3 to 10.sup.-4. Preferably, the method should be selective, such that the unwanted majority of clones are destroyed while the desirable rare clones survive.
Type II restriction-modification systems are being cloned with increasing frequency. The first cloned systems used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcORII:Koshyh, et al., Molec. Gen. Genet. 178:717-719 (1980); HhaII:Mann, et al., Gene 3:97-112 (1978); PstI:Walder, et al., Proc. Nat. Acad. Sci. USA 78:1503-1507 (1981)). Since the presence of restriction-modification systems in bacteria enable them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from libraries that have been exposed to phage. This method has been found, however, to have only limited value. Specifically, it has been shown that cloned restriction-modification genes do not always manifest sufficient phage resistance to confer selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning plasmids (EcoRV:Bougueleret, et al., Nucl. Acid Res. 12:3659-3676 (1984); PaeR7:Gingeras and Brooks, Proc. Nat. Acad. Sci. USA 80:402-406 (1983); Theriault and Roy, Gene 19:355-359 (1982): PvuII:Blumenthal, et al., J. Bacteriol. 164:501-509 (1985)).
A third approach, being used to clone a growing number of R-M systems, is by selection for an active methylase gene (refer to EPO No. 193 413 published Sep. 3, 1986 and BsuRI:Kiss, et al., Nucl. Acad Res. 13:6403-6421 (1985)). Since restriction and modification genes are often closely linked, both genes 10 can often be cloned simultaneously. This selection does not always yield a complete restriction system, however, but instead yields only the methylase gene (BspRI:Szomolanyi, et al., Gene 10:219-225 (1980); BcnI:Janulaitis, et al., Gene 20:197-204 (1982); BsuRI:Kiss and Baldauf, Gene 21:111-119 (1983); and MspI:Walder, et al., J. Biol. Chem. 258:1235-1241 (1983)).
A more recent method (the "Endo-Blue" method) has been described for direct cloning of restriction endonuclease genes using an indicator strain of E. coli containing a dinD::lacZ fusion. This method utilizes the E. coli SOS response following DNA damages by endonuclease or non-specific nucleases. A number of thermostable nuclease genes (BsoBI, TaqI, Tth111I, Tf nuclease) have been cloned using this method (Fomenkov, et al., Nucl. Acid Res. 22:2399-2403 (1994)).
Another obstacle to cloning these genes in E. coli was discovered in the process of cloning diverse methylases. Many E. coli strains (including those normally used in cloning) have methylation-dependent restriction systems (McrA, McrBC and Mrr) that resist the introduction of DNA containing methylated cytosine or adenine bases (Raleigh and Wilson, Proc. Nat. Acad. Sci. USA 83:9070-9074 (1986); Heitman and Model, J. Bact. 169:3243-3250 (1987)). Therefore, it is also necessary to carefully consider which E. coli strain(s) to use for cloning methylase genes.
Because purified restriction endonucleases, and to a lesser extent, modification methylases, are useful tools for characterizing genes in the laboratory, there is a commercial incentive to obtain bacterial strains through recombinant DNA techniques that synthesizes these enzymes in abundance. Such strains would be useful because they would simplify the task of purification, as well as providing the means for production of these enzymes in commercially useful amounts.